Fluorene-Based Fluorometric and Colorimetric Conjugated Polymers for Sensitive Detection of 2,4,6-Trinitrophenol Explosive in Aqueous Medium

Nitroaromatic explosives are a class of compounds that are responsible for various health hazards and terrorist outrages. Among these, sensitive detection of 2,4,6-trinitrophenol (TNP) explosive has always been highly desirable considering public health and national security. In this regard, three fluorene-based conjugated polymers (CP 1, CP 2, and CP 3) were synthesized through the Suzuki–Miyaura coupling reaction and were found to be highly sensitive for fluorescence detection of TNP with detection limits of 3.2, 5.7, and 6.1 pM, respectively. Excellent selectivity of CPs toward TNP was attributed to their unique π-π interactions based on fluorescence studies and density functional theory (DFT) calculations. The high sensitivity of CPs to TNP was attributed to the static quenching mechanism based on the photoinduced electron transfer process and was evaluated by fluorescence, UV–visible absorption, dynamic light scattering, Job’s plots, the Benesi–Hildebrand plots, and DFT calculations. CPs were also used for colorimetric and real-water sample analysis for the detection of TNP explosive. Meanwhile, sensor-coated test strips were fabricated for on-site detection of TNP, which makes them convenient solid-supported sensors.


SI-1. Materials and Methods
All chemicals and reagents such as 2,7-dibromo-9H-fluorene, n-octyl bromide, sodium carbonate, having a combination of halogen and deuterium lamp with fixed spectral bandwidth was used for UV-Vis analysis.
Progress of reaction was monitored by thin layer chromatography. Reaction mixture was slowly cooled down to room temperature. The desired product 5 was extracted with dichloromethane and washed with brine. The organic layer was collected and then dried over anhydrous MgSO4. The solvent of organic layer was removed under reduced pressure. The crude solid material was further chromatographed by silica gel column chromatography using n-hexane/petroleum ether as an eluent. The fluorene analogue 5 was obtained in 85% yield as yellow powder. temperature and continued to stir for 5 minutes. Then KI (0.7 mmol, 0.11 g) was added to the reaction mixture. After 5 minutes of stirring n-octyl bromide 3 was added to reaction and the mixture was heated at same temperature for 15 hours. Reaction mixture was allowed to cool down to room temperature. Product 6 was extracted from reaction mixture with dichloromethane and washed with brine and water for several times. Extracted organic layer was dried over MgSO4.
Solvent from organic layer was removed under reduced pressure. The crude oily liquid was further chromatographed by column chromatography using n-hexane as an eluent. Desired product 6 was obtained in 90% yield as white pale yellow liquid. S5
2,7-Dibromofluorene 1 (0.97 g, 3.0 mmol) was dissolved in 8 mL of ethanol at reflux. Afterward, potassium tertiary butoxide (0.37 g, 3.3 mmol) was added to reaction mixture. After an hour of reflux cinnamaldehyde 4 (0.39 g, 3.0 mmol) was slowly added to reaction mixture under inert atmosphere conditions. The color of the solution was instantly changed into a yellow solution.
Reaction timing was optimized through TLC analysis. Resulting reaction mixture was continued for further reflux until the full consumption of corresponding fluorene analogue 1. The desired product 7 was extracted with dichloromethane and washed with brine and distilled with water twice. Extracted organic layer was first dried over anhydrous MgSO4. Solvent of organic layer was removed by using rotary evaporator. The crude solid material was further chromatographed by silica gel column chromatography using n-hexane/petroleum ether as eluent. The desired product 7 was obtained in 70% yield as a yellowish powder.

SI-2.6. Procedure for Synthesis of Fluorene-Based Polymer (CP 3)
2,7-Dibromo-9-(3-phenylidene)9H-fluorene 7 (1.0 mmol, 1.7 g) was dissolved in 16 mL of THF at a temperature of 50 0 C under inert conditions. Afterward, Pd(PPh3)4 (0.03 mmol, 0.04 g) was added to reaction mixture followed by the addition of 8 mL of aqueous potassium carbonate (2.0 M) and 1,4-phenylenediboronic acid 8 (2.2 mmol, 0.36 g). Subsequently, whole of this reaction mixture was stirred for 36 hours at 85 °C. After this reaction mixture was cooled down to room temperature and diluted with 100 mL of chloroform. The collected organic layer was dried over anhydrous MgSO4 and solvent was removed by rotary evaporator. The crude was dissolved in 1 mL of chloroform and poured in 100 mL of methanol. Subsequently, precipitates were filtered off.

S7
The crude product was dissolved in 1.0 mL of chloroform and precipitated in methanol twice to obtain a brown solid with 81% yield.

SI-3. Fluorescence Spectral Acquisition
Fluorescence based sensing evaluations were carried out for various nitroaromatic compounds

SI-4. Determination of Association Constant (ka)
Binding efficiency confirms the strong association between conjugated polymer and analyte. This binding efficiency was calculated using the following Benesi-Hildebrand equation Where FX, Flim, and F0 represent the emission intensity of a CP at a medium concentration of nitroaromatic compounds, fluorescence intensity at the maximum association between CP and an analyte, and intensity of fluorescent compound, respectively. While C is the analyte concentration, n is the number of analytes being sensed by the sensor (here, n=1) and K is the binding or association constant (ka).

S8
Limit of detection (LOD) was estimated through the following equation 2.

SI-6. Job Plot Measurement
Binding stoichiometry of a conjugated polymer and nitroaromatic compounds was estimated by continuous variation method that is regarded as Job's plot. To obtain Job's plot measurement (continuous variation measurement), 10 µM of CP 1, CP 2, and CP 3 was taken and the mole fraction of respective analyte was gradually added to it in aqueous media H2O/THF (3:2, v/v).
Relative fluorescence intensity (I-I0) was plotted against mole fractions of analyte to obtain Job's plot.

SI-7. Determination of Percent Quenching Efficiency
The percent quenching efficiency was calculated from gradual decrease in emission intensity of conjugated polymer by increasing the concentration of nitroaromatic compounds. The following equation was applied to calculate the percent quenching efficiency of conjugated polymer. Where, I is emission intensity of sensor with maximum concentration of an analyte and I0 represents the emission intensity of pure conjugated polymer without any analyte.

SI-8. Computational methods
Theoretical studies via density functional theory (DFT) were executed by the Gaussian 09 program 2 and their graphical results were assessed by using GaussView 5.0 program. 3 The most stable geometries were obtained through optimization and frequency calculations at B3LYP/6-31G(d) method, which is considered as a suitable choice to investigate the geometric, thermodynamic, and electronic properties. 4 Interaction potential between CP 1, CP 2, and CP 3 and various analytes were calculated through equation 4. Interaction mechanisms between conjugated polymer and analytes were visualized through HOMO and LUMO analysis, performed at same level of DFT.

S9
Changes in HOMO-LUMO energy gap (H-L gap) provided deeper insight into their interactions which can be calculated through equation 5.
Isosurfaces of HOMO and LUMO orbitals were drawn through Gnuplot 4.6.5. 5

SI.10. Detection of Explosives in Industrial Effluents
Industrial wastewater samples were collected from Pakistan Ordinance Factory (POF), Havelian, Abbottabad, KPK, Pakistan. Impurities like dust and other small particles were removed through filtration using Whatman filter paper. The water samples were spiked with TNP (10 and 50 PM) to test determination and recovery of these nitroaromatic explosives. Same experimental parameters were employed for the fluorescence detection of TNP in real samples.

SI-11. Preparation of Sensors' Coated Test Strips
The TLC plates (thickness: 250 μm) were utilized for the construction of explosive responsive sensors' coated test strips. The TLC plates were immersed in 10 μM solution of CP 1, CP 2, and CP 3 in THF. Immersed paper strips were used for direct sensing after drying in an oven at 30 °C for 1 hour. TNP (10 nM) were applied onto the dry TLC strips adsorbed with respective conjugated polymer.